Two critically important areas in oncology are (i) the development of effective treatments that minimize damage to normal tissue, and (ii) the development of treatment strategies that are successful in reducing or eliminating metastatic disease. Cancer chemotherapy can inflict severe damage to normal tissue resulting in devastating side-effects. Metastatic disease is usually refractory to treatment and is a major cause of patients succumbing to cancer. The ability to downregulate specific pathways that are overexpressed and critically important to cancer cells using small interfering RNA (siRNA) technology provides unprecedented opportunities to develop novel cancer-cell specific treatments to target primary and metastatic tumors. The ability to detect the delivery of the siRNA and combine it with the delivery of a chemotherapeutic agent primarily localized within the tumor would be of significant advantage in this quest. We recently developed a prototype agent that allows us to visualize the delivery of a prodrug enzyme, bacterial cytosine deaminase, noninvasively with MRI and optical imaging. With this ability it is possible to time the administration of the nontoxic prodrug 5-fluorocytosine, that is converted by the enzyme to cytotoxic 5-fluorouracil, to coincide with the presence of high concentrations of the enzyme in the tumor and low concentrations in normal tissue, to minimize systemic toxicity. We intend to advance the effectiveness of this strategy by incorporating siRNA targeting of two enzymes, choline kinase and cyclooxygenase-2, that are critically important in breast cancer. The purpose of this application is two-fold. The first is to develop effective cancer treatment strategies utilizing imaged guided prodrug enzyme-siRNA treatment to minimize damage to normal tissue, using preclinical models of breast cancer. The second is to use these strategies, preclinically, to target metastatic lesions. Both MR and optical reporter systems are included in these prototype agents since MR is for clinical translation, whereas the optical reporter is useful for visualizing the incorporation of the enzyme-siRNA conjugate with high-resolution microscopy. The studies proposed in this application can also, in the future, be extended to image-guided targeting of radiation or chemotherapy repair enzymes in combination with prodrug enzyme treatment and radiation therapy. PUBLIC HEALTH RELEVANCE: Our purpose in this application is two-fold. The first is to develop effective treatment strategies utilizing imaged guided prodrug enzyme-siRNA treatment that will minimize damage to normal tissue, thereby reducing or eliminating the damaging side-effects of cancer treatment. The second is to use these strategies to target metastatic lesions, as breast cancer metastasis is refractory to treatment and a major cause of mortality, and there is an urgent need to identify new treatments that are effective at reducing or eliminating metastasis.